The Na/K-ATPase (sodium-pump; a receptor for cardiac glycosides, such as ouabain) is a multi-subunit integral membrane protein whose function (ATP-dependent transport of Na+ and K+ ions) is essential in maintenance and regulation of cardiac functions. Under the previous support, we identified the ouabain-binding region, Na- and K-sensitive segments, and the subunit assembly domain, and proposed a 'functional domain model' of the sodium-pump alpha subunit. Elucidation of the spatial organization of these functional domains is essential for understanding the molecular mechanism of the sodium-pump function. We have started to use a recently developed physical technique, atomic force microscopy (AFM) which as a few angstrom resolution, and identified a channel-like structure of the pump molecule. This is the first example that AFM technique brought in new information to biomedical sciences. On the basis of our new information and technical skills, we propose to identify the roles of Na+- and K+-sensitive domains (ion sensors) of the alpha subunit and elucidate the spatial organization of these functional domains. This goal will be attained by accomplishing the following specific aims: Aim 1: to define the regulatory roles of ion-sensitive domains in the Na/K- ATPase alpha-subunit (the amino-terminal 69 amino acids (Met1-Leu69) for Na+ and the carboxy-terminal 161 amino acids (Ser830-COOH) for K+), and to identify critical domains for ion-transport. Aim 2: to correlate the primary structure to the higher-order structures. Aim 1 will be addressed by using recombinant DNA and gene transfer techniques. Using all the sodium-pump/calcium-pump chimeras constructed in the previous support years as well as a new set of chimeras expressed in tissue- cultured cells and Xenopus oocytes, we will examine the roles of the ion- sensitive domains and search for the critical domains required for ion transport. Aim 2 will be addressed by using the techniques in structural biology. Over the past four years, we have developed specimen preparation techniques for the use of AFM, and now propose to correlate the 'channel-like' structure to a distinct conformation of the sodium- pump by comparing well-characterized biochemical effects of known ions and toxins on the sodium-pump structure. We plan to use electron microscopy (EM) as a comparative standard and for general analyses of the protein preparations throughout this proposal, although EM provides lower resolutions. Accomplishments of these aims will answer the following fundamental questions towards understanding the molecular structure- function relationship of ion pumps in general: "How do the Na+- and the K+-sensors of the alpha subunit regulate the sodium-pump activity?", "Which domains of the alpha subunit are essential for Na+ and/or K+ translocation?", and "How do ions and inhibitors modify the 'channel- like' conformation of the sodium-pump protein?". The proposed approaches also have a potential to detect conformational changes of the P-type- ATPase, and will be the first opportunity to visualize actual changes in the molecular structure of ion pumps.